Project Summary Most known genetic disorders arise from germline mutations that are present in every cell of the body. However, somatic mutations that occur in neural progenitors during brain development are increasingly recognized as a cause of focal brain malformations and epilepsy. Identifying mutations that cause these focal malformations is extremely challenging because (1) brain tissue from patients must be available and (2) on average only about 5% or fewer of cells in resected cortical tissue contain the causative mutation. For this reason, targeted sequencing of genes expected to cause focal cortical dysplasia (FCD) has been the only successful method for gene identification, and the yield is often very low. We hypothesize that an unbiased cell culture-based, high-throughput screening approach will identify many novel FCD genes. To identify these unknown genes, in Aim 1 we will use a CRISPR interference (CRISPRi) library screen to multiplex knockdown of nearly every gene in cultured human induced pluripotent stem cell (iPSC)-derived, developing excitatory cortical neurons. Positive hits will be isolated by fluorescence-activated cell sorting (FACS) for elevated phopho-S6 (and other dysplastic cell markers) and identified by next-gen sequencing. This screen will allow us to determine which genes, when turned off, lead to FCD-like human brain cell characteristics. In Aim 2, we will perform multiplexed CRISPRi, single-cell RNA-sequencing (direct capture Perturb-seq) for all of the candidate genes found in Aim 1, as well as for all known FCD genes, to both confirm the knockdown of the target gene and compare the transcriptomic effects of each FCD gene candidate. By comparing with known FCD genes, we will identify candidate genes with high confidence and construct an FCD transcriptomic ?fingerprint? that can be used as a resource for further understanding disease mechanisms. In Aim 3, we will apply focal CRISPRi knockdown of DEPDC5, a known FCD gene, to cortical organoids as an in vitro human FCD model. By combining these cutting-edge methods - high-throughput CRISPRi genetic screening, multiplexed gene expression fingerprinting, and brain organoid cultures - these studies will advance our understanding of FCD genetic causes and pathophysiology, providing a platform for future therapeutic studies.